1. Field of the Invention
The present invention is directed generally to the reaction that occurs between glucose and proteins, and more specifically to the identification of the reaction between reducing sugars and amino acids or proteins with nuclear material (DNA), and the application of this reaction to the synthesis of proteins of altered structure.
2. Description of the Prior Art
The reaction between glucose and proteins has been known for some time. Its earliest manifestation was in the appearance of brown pigments during the cooking of food, which was identified by Maillard in 1912, who observed that glucose or other reducing sugars react with amino acid to form adducts that undergo a series of dehydrations and rearrangements to form stable brown pigments. Maillard, L. C. (1912) C.R. Acad. Sci., Vol. 154, pp. 66-68.
In the years that followed the initial discovery by Maillard, food chemists studied the hypothesized reaction in detail and determined that stored and heat treated foods undergo nonenzymatic browning as a result of the reaction between glucose and the polypeptide chain, and that the proteins are resultingly crosslinked and correspondingly exhibit decreased bioavailability. Finot, P. A. (1982) in Modification of Proteins, eds, Feeney, R. E. and Whitaker, J. R., American Chemical Society, Vol. 198, pp. 91-124, Washington, D.C. At this point, it was determined that the pigments responsible for the development of the brown color that develops as a result of protein glycosylation possessed characteristic spectral properties; however, the chemical structure of the pigments had not been specifically elucidated.
The reaction between reducing sugars and proteins discussed above was found in recent years to have its parallel in vivo. Thus, the nonenzymatic reaction between glucose and the free amino groups on proteins to form a stable 1-amino-1-deoxy-2-ketosyl adduct, known as the Amadori product, has been shown to occur with hemoglobin, wherein a rearrangement of the amino terminal of the beta-chain of hemoglobin by reaction with glucose, forms the adduct known as hemoglobin A.sub.1c. This reaction was also found to occur with a variety of other body proteins, such as lens crystallins, collagen and nerve proteins. See, Bunn, H. F., Haney, D. N., Gabbay, K. H. and Gallop, P. H. (1975) Biochem. Biophys. Res. Comm. Vol. 67, pp. 103-109; Koenig, R. J., Blobstein, S. H. and Cerami, A. (1977) J. Biol. Chem. Vol. 252, pp. 2992-2997; Monnier, V. M. and Cerami, A. (1983) in Maillard Reaction in Food and Nutrition, ed. Waller, G. A., American Chemical Society, Vol. 215, pp. 431-448; and Monnier, V. M. and Cerami, A., (1982) Clinics in Endocrinology and Metabolism Vol. 11, pp. 431-452. Moreover, brown pigments with spectral and fluorescent properties similar to those of late-stage Maillard products have also been observed in vivo in association with several long-lived proteins, such as lens proteins and collagen from aged individuals. An age related linear increase in pigment was observed in human dura collagen between the ages of 20 to 90 years. See, Monnier, V. M. and Cerami, A. (1983) Biochem. Biophys. Acta., Vol. 760, 97-103 (1983); and, Monnier, V. M., Kohn, R. R. and Cerami, A. "Accelerated Age-Related Browning of Human Collagen in Diabetes Mellitus", (1983) Proc. Nat. Acad Sci. 81, 583-7. Interestingly, the aging of collagen can be mimicked in vitro by the crosslinking induced by glucose; and the capture of other proteins and the formation of adducts by collagen, also noted, is theorized to occur by a crosslinking reaction, and is believed to account for the observed accumulation of albumin and antibodies in kidney basement membrane. See, Brownlee, M., Pongor, S. and Cerami, A. (1983) J. Exp. Med., 158, 1739-1744 (1983).
As a result of recent studies by one of the inventors herein, further information regarding the chemistry of the late-stage Maillard process has been elucidated, and in particular, certain compounds have been identified that reflect the commencement and existence of the nonenzymatic reaction between proteins and glucose.
Specifically, in patent application Ser. No. 590,820, now U.S. Pat. No. 4,665,192, and in Ser. No. 097,856, filed Sep. 17, 1987, the disclosures of which are both incorporated herein by reference, certain chromophores reflecting these reactions were isolated and found to exist. Of the chromophores identified, the second was found to exist in instances where sulfite inhibition of the Maillard reaction had taken place. The existence of both chromophores confirmed generally the occurrence of nonenzymatic glycosylation of proteins, and specifically, the occurrence of advanced nonenzymatic glycosylation, and prompted further investigations as to the extent of protein-glucose interactions of this kind.
In this connection, the present inventors investigated the possibility that a similar nonenzymatic glycosylation could take place with genetic material encoding cellular proteins leading to a specific alteration of gene structure. This was prompted inasmuch as the earlier work referenced above had revealed that the Maillard reaction was taking place with several long-lived macromolecules in vivo.
Likewise, certain preliminary investigations by Bucala et al., PROC. NATL. ACAD. SCI. U.S.A. (1984), vol. 1981, pp. 105-109, and (1985), vol. 1982, pp. 8439-8442, investigated the effect of glucose 6-phosphate in causing the nonenzymatic modification of DNA and other nucleotides as measured by changes in spectral and fluorescent properties which were similar to those of other nonenzymatically glycosylated proteins. Following incubation of phage or plasmid DNA with glucose 6-phosphate, decreases in transfection and transformation capacities, respectively, were observed. These reactions appear to have a mutagenic effect, in some instances, resulting in both insertions and deletions in the plasmid DNA sequence and the development of multiple plasmid species from a single transformed cell. These earlier findings, however, were inconclusive as to the exact connection or causation for such phenomena, as it was observed by the investigators that certain of the aberrations noted were possibly attributable to glucose 6-phosphate reaction, however were also expected in the instance where DNA is exposed to other agents that may damage it.
Although both groups of investigators speculated respecting the implications of the glucose 6-phosphate reaction with nucleic acid material, none were certain of the mechanism of operation. Further investigations performed by the inventors herein have revealed additional information respecting the glycosylation of nucleic acids and the consequences thereof that are believed particularly relevant to clarifying the aging process as applied to these molecules and forming the basis for further investigations thereof as well as potential diagnostic and therapeutic applications. It is to these latter aims the present invention is accordingly directed.